1. 湖南中医药大学研究生院
2. 湖南中医药大学医学院
3. 湖南中医药大学中医学院
纸质出版:2017
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[1]饶春梅,成细华,任婷,孙彦波,彭霞,黄政德.加味丹参饮含药血清对缺氧/复氧H9C2心肌细胞自噬相关蛋白LC3Ⅱ及Beclin1表达的影响[J].中医杂志,2017,58(12):1043-1048.
RAO Chunmei, CHENG Xihua, REN Ting, et al. Impact of Serum Containing Jiawei Danshen Yin( 加味丹参饮) on Expressions of Hypoxia/Reoxygenation H9C2 Cardiac Muscle Cell Autophagy Related Protein LC3Ⅱ and Beclin1[J]. Journal of Traditional Chinese Medicine2017, 58(12): 1043-1048.
[1]饶春梅,成细华,任婷,孙彦波,彭霞,黄政德.加味丹参饮含药血清对缺氧/复氧H9C2心肌细胞自噬相关蛋白LC3Ⅱ及Beclin1表达的影响[J].中医杂志,2017,58(12):1043-1048. DOI: 10.13288/j.11-2166/r.2017.12.015.
RAO Chunmei, CHENG Xihua, REN Ting, et al. Impact of Serum Containing Jiawei Danshen Yin( 加味丹参饮) on Expressions of Hypoxia/Reoxygenation H9C2 Cardiac Muscle Cell Autophagy Related Protein LC3Ⅱ and Beclin1[J]. Journal of Traditional Chinese Medicine2017, 58(12): 1043-1048. DOI: 10.13288/j.11-2166/r.2017.12.015.
目的探讨加味丹参饮治疗心肌缺血再灌注损伤的可能作用机制。方法将体外培养的H9C2心肌细胞随机分为6组
每组6个复孔。空白血清组在含15%空白血清培养液中常规培养29 h;缺氧预处理组更换缺氧培养液缺氧20 min
复氧20 min
再缺氧20 min后换为空白血清培养液常规培养24 h
缺氧2 h
再给氧3 h;缺氧/复氧组在含15%空白血清培养液中常规培养24 h
更换缺氧培养液
缺氧2 h
再给氧3 h;含药血清组在含15%药物血清中常规培养24 h
缺氧2 h
再给氧3 h;含药血清+自噬抑制剂组用3-甲基腺嘌呤10 mmol/L预处理30 min
余同含药血清组;含药血清+自噬激动剂组用雷帕霉素100 nmol/L预处理30 min
余同含药血清组。倒置显微镜观察各组心肌细胞生长及形态变化
比色法检测定各组细胞培养上清液中乳酸脱氢酶(LDH)漏出率
透射电子显微镜观察心肌细胞自噬体形态及数量变化
Western blot法检测各组自噬相关蛋白LC3Ⅱ及Beclin1表达。结果与空白血清组比较
缺氧/复氧组镜下心肌细胞变性坏死程度增加
电镜下有少量自噬体
培养液中LDH漏出率增加
Beclin1蛋白水平增加(P<0.05或P<0.01);与缺氧/复氧组比较
含药血清组及含药血清+自噬激动剂组镜下心肌细胞变性坏死程度减轻
电镜下自噬体数量增多
培养液中LDH漏出率显著降低
LC3Ⅱ及Beclin1蛋白表达上升(P<0.05)。结论加味丹参饮可能通过上调自噬相关蛋白LC3Ⅱ及Beclin1表达
增强细胞自噬
从而保护损伤的心肌细胞。
Objective To explore the possible mechanism of Jiawei Danshen Yin( 加味丹参饮) in treating cardiac reperfusion injury. Methods H9C2 cardiac muscle cells in vitro were randomized into 6 groups
with 6 multiple pores in each group. The blank serum group was cultured in culture medium containing 15% of blank serum for 29 h.The hypoxia preconditioning group was cultured in hypoxia medium for 20 min
and then was given oxygen for 20 min
following another hypoxia culture for 20 min. After then the cells were moved into blank serum medium and routinely cultured for 24 h
following cultured in hypoxic medium for 2 h
and then given oxygen for 3 h. The hypoxia/reoxygenation group was cultured in culture medium containing 15% of blank serum for 24 h
then was moved into hypoxia medium and cultured in hypoxia condition for 2 h
then in oxygenation condition for 3 h. The serum containing medicine group was cultured in culture medium containing 15% of medicine serum in normal condition for 24 h
and then was in hypoxia condition for 2 h
following in oxygenation condition for another 3 h. The serum containing medicine + autophagy inhibitor group was preconditioned by 10mmol/L 3-methyladenine for 30 min; the following treatment steps were the same as the serum containing medicine group. The serum containing medicine + autophagy agonist group was preconditioned by 100 nmol/L rapamycin for 30 min; the other treatment steps were the same as the serum containing medicine group. Growth and form changes of cardiac muscle cells in each group were examined with inverted microscope. The leakage rate of lactate dehydrogenase( LDH) in cell culture supernatant in each group was detected with colorimetric method. The form and number changes of myocardial cell autophagosome were examined with transmission electron microscope. The expressions of autophagy related protein LC3 Ⅱ and Beclin1 in each group were detected with Western blot method. Results Compared with that in the blank serum group
the degree of degeneration and necrosis of myocardial cells increased under microscope
there were a few autophagosome under electron microscope
the leakage rate of LDH in medium increased
and the level of Beclin1 protein increased in the hypoxia/reoxygenation group( P < 0. 05 or P < 0. 01). Compared with the hypoxia/reoxygenation group
the degree of degeneration and necrosis of myocardial cells decreased
the number of autophagosome increased under electron microscope
the leakage rate of LDH in medium decreased obviously
and the expressions of LC3 Ⅱ and Beclin1 protein increased in the serum containing medicine group and the serum containing medicine + autophagy agonist group( P <0. 05). Conclusion Jiawei Danshen Yin might strengthen autophagy by up-regulate the expressions of autophagy related protein LC3Ⅱ and Beclin1 to protect injured cardiac muscle cells.
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